Mark D. Lindner

Implants of Encapsulated Human CNTF-Producing Fibroblasts Prevent Behavioral Deficits and Striatal Degeneration in a Rodent Model of Huntington’s Disease

Delivery of neurotrophic molecules to the CNS has gained considerable attention as a potential treatment strategy for neurological disorders. In the present study, a DHFR-based expression vector containing the human ciliary neurotrophic factor (hCNTF) was transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting hCNTF (BHK-hCNTF) were transplanted unilaterally into the rat lateral ventricle. Twelve days later, the same animals received unilateral injections of quinolinic acid (QA; 225 nmol) into the ipsilateral striatum. After surgery, animals were behaviorally tested for apomorphine-induced rotation behavior and for skilled forelimb function using the staircase test. Rats receiving BHK-hCNTF cells rotated significantly less than animals receiving BHK-control cells. No behavioral effects of hCNTF were observed on the staircase test. Nissl-stained sections demonstrated that BHK-hCNTF cells significantly reduced the extent of striatal damage produced by QA. Quantitative analysis of striatal neurons further demonstrated that both choline acetyltransferase- and GAD-immunoreactive neurons were protected by BHK-hCNTF implants. In contrast, a similar loss of NADPH-diaphorase-positive cells was observed in the striatum of both implant groups. Analysis of retrieved capsules revealed numerous viable and mitotically active BHK cells that continued to secrete hCNTF. These results support the concepts that implants of polymer-encapsulated hCNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage and that this strategy may ultimately prove relevant for the treatment of Huntington’s disease.

Incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine produce akinesia, rigidity, tremor and cognitive deficits in middle-aged rats.

The present study was conducted to determine if the full array of parkinsonian symptoms could be detected in rats with nigrostriatal cell loss and striatal dopamine depletions similar to levels reported in the clinical setting, and to determine if older rats exhibit more robust parkinsonian deficits than younger rats. Young (2 months old) and middle-aged (12 months old) rats received bilateral striatal infusions of 6-OHDA, over the next 3 months they were assessed with a battery of behavioral tests, and then dopaminergic nigrostriatal cells and striatal dopamine and DOPAC levels were quantified. The results of the present study suggest that: (1) the full array of parkinsonian symptoms (i.e. akinesia, rigidity, tremor and visuospatial cognitive deficits) can be quantified in rats with incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine levels (2) parkinsonian symptoms were more evident in middle-aged rats with 6-OHDA infusions, and (3) there was evidence of substantial neuroplasticity in the older rats, but regardless of the age of the animal, endogenous compensatory mechanisms were unable to maintain striatal dopamine levels after rapid, lesion-induced nigrostriatal cell loss. These results suggest that using older rats with nigrostriatal dopaminergic cell loss and reductions in striatal dopamine levels similar to those in the clinical condition, and measuring behavioral deficits analogous to parkinsonian symptoms, might increase the predictive validity of pre-clinical rodent models.

Cellular delivery of human CNTF prevents motor and cognitive dysfunction in a rodent model of Huntington's disease

The delivery of ciliary neurotrophic factor (CNTF) to the central nervous system has recently been proposed as a potential means of halting or slowing the neural degeneration associated with Huntingtons disease (HD). The following set of experiments examined, in detail, the ability of human CNTF (hCNTF) to prevent the onset of behavioral dysfunction in a rodent model of HD. A DHFR-based expression vector containing the hCNTF gene was transfected into a baby hamster kidney fibroblast cell line (BHK). Using a polymeric device, encapsulated BHK-control cells and those secreting hCNTF were transplanted bilaterally into rat lateral ventricles. Eight days later, the same animals received bilateral injections of quinolinic acid (QA, 225 nmol) into the previously implanted striata. A third group received sham surgery (incision only) and served as a normal control group. Bilateral infusions of QA produced a significant loss of body weight and mortality that was prevented by prior implantation with hCNTF-secreting cells. Moreover, QA produced a marked hyperactivity, an inability to use the forelimbs to retrieve food pellets in a staircase test, increased the latency of the rats to remove adhesive stimuli from their paws, and decreased the number of steps taken in a bracing test that assessed motor rigidity. Finally, the QA-infused animals were impaired in tests of cognitive function-the Morris water maze spatial learning task, and the delayed nonmatching-to-position operant test of working memory. Prior implantation with hCNTF-secreting cells prevented the onset of all the above deficits such that implanted animals were nondistinguishable from sham-lesioned controls. At the conclusion of behavioral testing, 19 days following QA, the animals were sacrificed for neurochemical determination of striatal choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) levels. This analysis revealed that QA decreased striatal ChAT levels by 35% and striatal GAD levels by 45%. In contrast, hCNTF-treated animals did not exhibit any decrease in ChAT levels and only a 10% decrease in GAD levels. These results support the concepts that implants of polymer-encapsulated hCNTF-releasing cells can be used to protect striatal neurons from excitotoxic damage, produce extensive behavioral protection as a result of that neuronal sparing, and that this strategy may ultimately prove relevant for the treatment of HD.

Rats with partial striatal dopamine depletions exhibit robust and long-lasting behavioral deficits in a simple fixed-ratio bar-pressing task

It is widely accepted that enduring parkinsonian symptoms are only evident if there are few remaining dopaminergic neurons in the substantia nigra and dopamine levels in the basal ganglia are very low [26,41]. In the present study, partial dopamine depletions were produced by infusing 6-OHDA bilaterally into the ventrolateral striatum as previously described [11,12,44]. Consistent with previous studies, behavioral deficits were detectable in rats with partial lesions with a simple fixed-ratio bar-pressing task. The present study demonstrated that these behavioral deficits were long-lasting, and that the sensitivity of this bar-pressing task could be increased by manipulating the level of difficulty of the task-higher fixed ratios were more sensitive to partial dopamine depletions. Deficits in rats with partial dopamine depletions could also be detected using non-automated neurological tests of parkinsonian symptoms developed for rats with severe unilateral dopamine depletions, but these deficits were transient and not as robust as those detected with the bar-pressing task. Oral Sinemet (L-DOPA:carbidopa) did not attenuate behavioral deficits related to partial dopamine depletions in this simple fixed-ratio bar-pressing task, but the present results suggest that Parkinsons patients might be identifiable earlier in the disease process, at a time when they could benefit from treatment with neuroprotective/neurotrophic agents. In addition, the results of the present study demonstrate that robust behavioral deficits may emerge with age. Mild dopamine depletions that were not detectable behaviorally at the time of the insult became clearly evident 10 months after the lesion with this bar-pressing task, and this may represent a more clinically relevant rodent model of Parkinsons disease.

Individual differences in the hotplate test and effects of habituation on sensitivity to morphine

&NA; Hotplate studies rarely match subjects into groups and often use high temperatures that are less sensitive to the effects of mild analgesics. Subjects may not be matched into groups because it has not been clearly demonstrated that there are reliable and robust individual differences in performance on the hotplate, and out of concern that the testing required to match subjects into groups might reduce the sensitivity of the task to mild analgesics by producing ‘behavioral tolerance’. Higher hotplate temperatures may be preferred because they reduce variability in response latencies, and it may be assumed that this precludes the need to match subjects into groups. The results of the present study demonstrate that there are reliable and robust differences among individuals tested on the hotplate, regardless of whether the hotplate is 50°C or 55°C (&agr;s > 0.90). The present results also confirm that lower hotplate temperatures are much more sensitive to the effects of mild analgesics: increased response latencies following a low dose of morphine (3 mg/kg) could be reliably detected with only 8 rats at 50°C, while the same dose would not be detected reliably at 55°C unless more than 55 rats were tested. Finally, there was no evidence that habituation to the hotplate produced ‘behavioral tolerance’ or reduced the sensitivity of the test to the effects of morphine. These findings suggest that hotplate studies should match subjects into groups and use lower hotplate temperatures in order to increase the sensitivity of the test, but also out of an ethical obligation to minimize the intensity of the noxious stimulus and the number of animals exposed to it.

Differential in vivo effects of neurturin and glial cell-line-derived neurotrophic factor.

Glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally homologous, and they seem to produce similar effects in vitro. Tissue distributions of their respective receptors, GFR alpha-1 and GFR alpha-2, reveal overlapping but distinct patterns of expression, which implies that the in vivo actions of GDNF and NTN may be different. In the present study, a direct comparison of the in vivo effects of GDNF and NTN was performed using osmotic minipumps delivering either GDNF or NTN over a 30-day period into rat lateral cerebral ventricles. Amphetamine-induced activity levels were increased in both NTN- and GDNF-treated animals, with higher activity levels achieved by GDNF than NTN. The increase in amphetamine-induced activity levels persisted for 2 weeks and returned to control levels at the end of the third week. NTN-treated rats showed higher dopamine levels in the mediodorsal striatum, relative to the ventrolateral striatum. In contrast, no significant change in the regional distribution of dopamine levels was observed in GDNF treated or control animals. On the other hand, an increase in ventrolateral and mediodorsal striatal dopamine utilization was apparent in GDNF-treated animals, while NTN-treated animals showed increased levels of dopamine utilization only in the ventrolateral striatum. With respect to potential adverse effects, GDNF administration resulted in weight loss and the emergence of allodynia. No weight loss or allodynia was detectable with chronic NTN administration. These results suggest that although GDNF and NTN share structural and functional similarities, they may have differential effects in vivo.

Therapeutic Potential of a Polymer-Encapsulated l-DOPA and Dopamine-Producing Cell Line in Rodent and Primate Models of Parkinson’s Disease

Encapsulation of cells within polymer membranes prior to transplantation provides a novel means of achieving continuous, site-specific delivery of therapeutic molecules to the CNS. The use of encapsulated dopamine-secreting cells that can be transplanted directly into the striatum has particular appeal for the treatment of Parkinsons disease. This article provides a brief and timely review of the progress that has been made over the past decade using encapsulated PC12 cells as a means of delivering dopamine and L-DOPA to the striatum in rodent and primate models of Parkinsons disease. The polymer membranes are well tolerated and biocompatible. Encapsulated PC12 cells survive in vivo for up to 6 mo, they release dopamine into the surrounding host striatum, and they clearly improve behavioral function in both dopamine-depleted rodents and primates. Although these results are promising, fundamental issues remain concerning the extent of dopamine diffusion from the polymer membranes and the number of devices needed for behavioral improvement, and the duration and consistency of cell viability and device output. Nevertheless, this technology appears to be a promising means of avoiding many of the practical, societal, and ethical issues that have been associated with other transplantation approaches.

Mammalian-cell-produced neurturin (NTN) is more potent than purified Escherichia coli-produced NTN

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor. Both factors promote the survival of dopaminergic (DA) neurons. We investigated the biological activity of mammalian-cell-produced NTN versus purified Escherichia coli-produced NTN. Baby hamster kidney cells were engineered to stably secrete mature human NTN. Mammalian-cell-derived NTN enhanced the activity of embryonic DA neurons in vitro, with greater potency (maximum effect achieved in the picogram range) than purified E. coli-produced NTN. Cell-based delivery of NTN (less than 10 ng/day) was also shown to be biologically active in vivo. These results suggest that mammalian-cell-derived NTN, synthesized de novo and delivered in small quantities to the parenchyma at the target site, may be as active as much larger quantities of purified, E. coli-produced NTN, delivered by other means.

Chronic morphine reduces pain-related disability in a rodent model of chronic, inflammatory pain.

Chronic pain is disabling, and the adverse effects of morphine are also disabling. The best way to assess the beneficial effects relative to the potential adverse effects of chronic morphine may be through the use of quantitative measures of functional disability in people and animals experiencing pain. If chronic morphine alleviates chronic pain and its beneficial analgesic effects outweigh whatever adverse effects it may produce, then it should reduce pain-related disability. Rats with adjuvant-induced arthritis were implanted with subcutaneous morphine pellets. Continuous morphine reduced pain-related disability in tasks motivated by food reward or shock avoidance throughout the 35 days of continuous administration--first, in tests that primarily assessed the function of the less severely affected forelimbs, and later, as the inflammation subsided, in tests more dependent on the function of the more severely affected hind limbs.

Numerous adrenal chromaffin cell preparations fail to produce analgesic effects in the formalin test or in tests of acute pain even with nicotine stimulation

&NA; Previous studies have reported that intrathecal implants of a variety of adrenal chromaffin cell preparations all produce analgesic effects in rodents. The major objective of the present study was to determine if any adrenal chromaffin cell preparations produce more robust analgesic effects than other cell preparations. The present study included adult rat adrenal chromaffin tissue allografts, purified adult bovine chromaffin cells, and polymer‐encapsulated calf adrenal chromaffin cells, all prepared according to previously published procedures, as well as purified calf adrenal chromaffin cells. Previous studies have also suggested that immunosuppression may play a role in graft survival, and potentially increase the magnitude of analgesic effects, so the present study included both immunosuppressed and non‐immunosuppressed groups (cyclosporin A, 10 mg/kg per day). Behavioral tests included the formalin test; and a dorsal tail‐flick, hot‐plate, and paw‐pinch test, conducted sequentially 2 min after systemic nicotine (0.1 mg/kg) to evoke release from the chromaffin cells, as previously reported. Analgesic effects related to morphine and nicotine were detected, and consistent differences in performance could be detected between individual animals. Surprisingly, no analgesic effects were detectable with any of the four chromaffin cell preparations, with or without immunosuppression, in the formalin test or with nicotine stimulation in tests of acute pain.